Method for dosing therapeutic compounds

ABSTRACT

The internally cyclized congeners of hydroxy-substituted nucleotide analogues have been found to exhibit substantially lower toxicity in vivo than their uncyclized analogues, while retaining essentially the same antiviral activity. This was unexpected because the prior art suggested that the cyclic analogues offered no significant advantages in respect to toxicity. This finding permits the administration of much greater doses of the cyclic congeners than otherwise would have been possible and allows the clinician to omit toxicity ameliorating interventions. Novel compounds are provided for use in the method of this invention. Novel methods for the preparation of these compounds also are provided.

This is a divisional of application Ser. No. 08/597,005 filed on Feb. 5,1996, now U.S. Pat. No. 5,591,851, which is a continuation of U.S. Ser.No. 08/193,341, filed Feb. 8, 1994, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 08/123,483, filed Sep. 17, 1993,now U.S. Pat. No. 5,656,745.

Cross-reference is made to related copending application U.S. Ser. No.08/612,889.

This invention relates to methods and compounds for the treatment ofviral infections, including prophylaxis. In particular it is concernedwith the management of kidney toxicity by selection of therapeuticdosages of antiviral compounds.

A number of antiviral compounds are known that are characterized by aphosphonate group linked to a nucleotide base via a hydroxy-substitutedcyclic or acyclic linking moiety, wherein the hydroxy group is joined by4 atoms (typically as alkyl or alkoxyalkyl chains) to the phosphorusatom and the phosphorus atom is bonded to a methylene group of thecyclic or acyclic linking moiety. These hydroxy-substituted nucleotideanalogues (herein, "HSNA"s) include the compounds of structures(I)-(VII) below. Structure (I) compounds are disclosed in EP 369,409and/or U.S. Ser. No. 08/110,841 (pending): ##STR1## wherein B is aheterocyclic group having at least 1 nitrogen heteroatom and up to 3additional heteroatoms selected from nitrogen, oxygen and sulfur, saidheterocyclic group being connected through a nitrogen heteroatomthereof, and R² is hydrogen, hydroxy, fluorine, chlorine, bromine,amino, or an organic substituent having 1-5 carbon atoms and selectedfrom acyloxy, alkoxy, alkylthio, alkylamino or dialkylamino.

Structure (II) and (III) compounds are disclosed in EP 398,231: ##STR2##wherein Z is hydrogen or C₁ -C₆ alkyl and B is a 9-substituted purine or1-substituted pyrimidine base; and ##STR3## wherein R' is hydrogen, C₁-C₆ alkyl or hydroxyalkyl with 1-6 carbon atoms, and B is a9-substituted purine or 1-substituted pyrimidine base.

Barnard et al. ("Antiviral Research" 22:77-89 1993!) disclose HSNAcompounds of structure (IV): ##STR4## wherein B is guanin-9-yl.

U.S. Pat. No. 5,208,221 discloses HSNA compounds of structure (V):##STR5## wherein B is a 9-substituted purine or 1-substituted pyrimidinebase.

U.S. Pat. No. 5,247,085 discloses HSNA compounds of structure (VI):##STR6## wherein R is H, C₁ -C₆ alkyl, or optionally substituted phenyland B is one of a group of defined purin-9-yl bases.

Other well-known HSNAs include certain3-hydroxy-2-(phosphonomethoxy)propyl analogues of nucleotide bases(herein, "HPMPB") of structure (VII): ##STR7## wherein B is apyrimidin-1-yl, pyrimidin-3-yl, purin-3-yl, purin-7-yl or purin-9-ylresidue, or the deaza, aza or deaza-aza analogues thereof. Thesecompounds are active against DNA viruses. The principal members of theHPMP class are the compounds of structure (VII) in which B iscytosin-1-yl (herein, "HPMPC") or adenin-9-yl (herein, "HPMPA"). The (S)isomers are preferred. See U.S. Pat. Nos. 5,142,051 and 4,724,233.

It is known to internally cyclize certain HSNA compounds. cHPMPBs arethe internally cyclized congeners of the corresponding HPMPB and havestructure (VIIa): ##STR8## wherein B is as defined in structure (VII).Two examples of the compounds of structure (VIIa) are known: cHPMPA andcHPMPC (U.S. Pat. No. 4,724,233 and Ho et al., "Mol. Pharmacology"41:197-202, 1992!). The (S) enantiomer of cHPMPC bears the IUPAC name 1-((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl! cytosine (CASReg. No. 127757-45-3).

In addition, the internally cyclized analogues of the compounds ofstructures (II) and (III) (EP 398,231) and (VI) (U.S. Pat. No.5,247,085) are known. They have the structures (IIa), (IIIa) and (VIa):##STR9## wherein Z, R, R' and B are defined above in structures (II),(III) and (VI).

HPMPC has been extensively studied and currently is in human clinicaltrials. Its cyclic congener has received comparatively little attention.However, cHPMPC has been reported to have activity againstcytomegalovirus in human embryonic lung cells in vitro (Snoeck et al.,"Antiviral Research" 16:1-9 1991!). The Snoeck et al. data suggest thatcHPMPC is less toxic than HPMPC, but less efficacious as well byapproximately the same degree. In particular, Snoeck et al. reportedthat the micromolar cytotoxicities of cHPMPC and HPMPC were 720 and 360,respectively, by cell growth and 108 and 72, respectively, byradiothymidine incorporation. Holy et al. ("Antiviral Research"13:295-312 1990!) reported similar in vitro cell culture data. See alsoSnoeck et al., "Int. Congr. Ser. Excerpta Med.," 978 (Prog.Cytomegalovirus Res.) 337-340 (1991) and Holy et al., "Coll. Czech.Chem. Commun." 54(a): 2470-2501 (1989).

Li et al. have reported that HPMPC is nephrotoxic in guinea pigs (see"Antiviral Research" 13:237-252 1990!), and nephrotoxicity is thelimiting toxicity in human clinical trials of HPMPC. Humannephrotoxicity is ameliorated by concomitant administration ofprobenecid and by giving fluids prior to HPMPC administration(hydration). In contrast to the extensive studies of HPMPC, thepublished literature is believed to be devoid of any animal studies ofefficacy or toxicity of cHPMPC.

It is an object of this invention to enlarge the therapeutic window forHSNAs by supplying them in a form that is less toxic in vivo whilesubstantially retaining the antiviral activity of the HSNA. In addition,it is an object of the invention to reduce or eliminate the practice ofhydration or probenecid administration during a course of HPMPC therapyand to minimize the need to withdraw patients from HSNA treatment due tothe development of kidney toxicity. An additional object of thisinvention is to facilitate non toxic increases in the dose, frequencyand length of administration of HSNAs. It is a further object of thisinvention to provide novel internally cyclized derivatives of certainHSNAs. Another object of this invention is to provide intermediate formsof HSNAs having oral bioavailability, reduced toxicity and greaterefficacy, together with novel methods for their manufacture.

SUMMARY OF THE INVENTION

The objects of this invention are accomplished by administering to asubject an antivirally effective, non-cytotoxic dose of cHSNA which isin excess of the maximum non-cytotoxic dose for the correspondinguncyclized HSNA. In certain embodiments the maximum non-cytotoxic doseis defined in terms of the maximum non-nephrotoxic dose. In preferredembodiments, particularly where the HSNA is HPMPC, the cHSNA dose is inexcess of 2 times the HSNA maximum non-cytotoxic dose. In additionalembodiments an antivirally effective course of therapy of cHPMPC orother cHSNA is administered without probenecid and/or hydration.

The cHSNAs to be used in the practice of this invention have structure(VIII) ##STR10## wherein Z₂ is oxygen or methylene, Y is --CH₂ --,--OCH₂ --, --O-- ##STR11## Z, R', and R² are defined above, A' is OH orA, A is an amidate or ester, the stereochemistry of the carbon andphosphorus atoms denoted with the asterisks independently are (S), (R)or (R,S), the orientation of the Y groups is shown by (B'), and B' is anunsubstituted nitrogen-, or nitrogen and sulfur-containing heterocyclicring structure or such ring structure substituted with from 1 to 3substituents independently selected from oxo, hydroxy, amino, fluoro,chloro, bromo, iodo, C₁ -C₉ haloalkyl (1-3 halo), C₁ -C₉ alkyl, C₂ -C₉alkenyl, C₂ -C₉ haloalkenyl, C₁ -C₉ alkoxy, C₂ -C₉ thioalkenyl, C₁ -C₉alkylthiol, amino C₁ -C₉ alkyl, amino C₃ -C₄ alkenyl, amino C₃ -C₄alkynyl, cycloamino C₂ -C₅ alkyl, thio C₁ -C₉ alkyl, C₁ -C₉hydroxyalkyl, C₁ -C₃ alkoxy, C₁ -C₄ alkyl, acylamine, thiol, =S, or=N--NH₂.

In other embodiments of the invention, novel compounds are providedwhich have the structures (Ia) and (Va): ##STR12## wherein * designates(S), (R) or (R,S) configuration, and B', R² and A' are defined above,together with the salts thereof.

Structure (Va) is ##STR13## wherein * and A' and B' are defined above,together with the salts thereof.

Also useful in this invention are compounds of structure (IVa) ##STR14##wherein * and A' and B' are defined above, together with the saltsthereof. In each of (Ia) and (Va), the carbon atom * chiral centerpreferably is (S); in (IVa) it preferably is (R).

This invention also includes the novel uncyclized counterparts of theforegoing cHSNAs of structure (VIII), which will differ from the knownuncyclized forms in the choice of base and/or choice of A' (the A'group(s) of the uncyclized counterparts will be OH, or monosubstitutedor disubstituted with A, wherein the A groups are the same ordifferent). Hereby excluded from the scope of this invention are anyuncyclized HSNAs that are not novel and unobvious over their prior artuncyclized counterparts.

DETAILED DESCRIPTION OF THE INVENTION

HPMPC is the HSNA for which the greatest human clinical experience isavailable. For the most part, HPMPC has been administered once or twiceweekly for 4 weeks, and patients completing the 4 week study withoutevidence of drug-related toxicity continued with weekly therapy.HIV-infected patients with asymptomatic CMV infection of urine and semenhave tolerated doses of HPMPC at 0.5, 1.0 and 1.5 mg/kg for 4 weekswithout evidence of significant HPMPC-related clinical or laboratorytoxicity.

Patients treated at the highest dose levels previously studied (5.0mg/kg twice weekly and 3.0 or 10.0 mg/kg once weekly) developed evidenceafter as few as 1 or 2 doses for HPMPC-related nephrotoxicity manifestedby proteinuria, glycosuria, and decreases in serum phosphate, uric acid,and bicarbonate consistent with renal proximal tubular cell injury. Twoof five patients receiving 3.0 mg/kg once weekly developed Grade IInephrotoxicity (serum creatinine of ≧2.0 mg/dL or 2+ proteinuria)following 6 and 14 doses of HPMPC, respectively. Two of five patientsreceiving 10.0 mg/kg once weekly of HPMPC developed evidence ofpersistent Grade IV nephrotoxicity following two doses. Both patientshad evidence of non-oliguric renal insufficiency consistent withproximal tubular cell injury. Each of the above four patients displayingnephrotoxicity did not receive concomitant hydration during theantecedent HPMPC infusions. Persistent nephrotoxicity (≧Grade II) hasnot been observed in patients receiving concomitant hydration withHPMPC.

Proteinuria, as measured by routine urinalysis, appears to be asensitive early indicator of HSNA-related nephrotoxicity. Interruptionof HPMPC treatment following the appearance of proteinuria has permittedthe administration of systemic HPMPC without significant drug-relatedtoxicity. Continued administration of HPMPC following the demonstrationof proteinuria with or without serum creatinine elevation can result inprolonged and potentially irreversible renal insufficiency.

Identification of the sequence of urinalysis and serum chemistryabnormalities associated with HPMPC-related nephrotoxicity, as well asdemonstration of prolonged anti-CMV effect, has led to modifications inthe methods of HPMPC administration (Table 1). For example, interruptionof HPMPC treatment following the appearance of ≧1+ proteinuria haspermitted the administration of systemic HPMPC without significantdrug-related toxicity. Investigation of longer dosing intervals (one,two, and three weeks) has also been pursued. Additionally, as suggestedby preclinical animal studies, concomitant administration of probenecidhas been employed in an effort to block uptake of HPMPC by the proximaltubular cell of the kidney.

                  TABLE 1                                                         ______________________________________                                        Phase I/II HPMPC Regimens:                                                    Dose - Refinements                                                            Dose (mg/kg)    Schedule Hydration.sup.1                                      ______________________________________                                        3.0 + Probenecid.sup.2                                                                        q week   +/-                                                  5.0 + Probenecid                                                                              q week   +/-                                                  5.0 + Probenecid                                                                              q 2 weeks                                                                              +/-                                                  7.5 + Probenecid                                                                              q 3 weeks                                                                              +/-                                                  ______________________________________                                         .sup.1 Administered as one liter normal saline over approximately 45          minutes immediately prior to HPMPC infusion.                                  .sup.2 Administered orally as 2 grams (3 h preHPMPC), 1 gram (2 h             postHPMPC), and 1 gram (8 h postHPMPC) (total dose = 4 grams).           

To date, 21 patients have received HPMPC with concomitant probenecid (8patients at 3 mg/kg range 2-11 doses!; 11 patients at 5 mg/kg range 2-8doses!; and 2 patients at 7.5 mg/kg range 1-2 doses!). None of thesepatients have developed proteinuria.

Three of 21 patients developed evidence of allergic symptoms temporallyrelated to probenecid administration. Each occurred after 3 to 4consecutive weeks of treatment. Two of three developed pruriticmaculopapular rashes responsive to antihistamine therapy, permittingcontinued administration. The third patient developed evidence of asystemic reaction including rash, nausea and headache. The contributionof other medications interacting with probenecid is uncertain at thistime; however, as zidovudine (AZT) levels have been demonstrated toincrease significantly when administered with probenecid, patients havebeen cautioned to withhold or reduce (e.g., 50 percent reduction) theirAZT doses on days of probenecid administration. Thus, it is desirable toeliminate the use of probenecid in treatment with HPMPC.

The serious adverse events observed in these clinical studies are listedbelow (Table 2). The table includes all reported serious adverse events,whether or not they were felt to be related to HPMPC. As noted above,nephrotoxicity appears to be the major dose-limiting toxicity related toHPMPC administration. Additionally, neutropenia has been observed inthese studies. It is noteworthy that this hematologic toxicity does notappear to be dose dependent. The frequency of neutropenia appears to becomparable with reported rates of neutropenia observed in similarpatient populations studied in controlled clinical trials (i.e.,patients with advanced HIV infection and CD4 cell count <100 cells/mm³receiving concomitant antiretroviral therapies).

                  TABLE 2                                                         ______________________________________                                        Serious Adverse Events Associated with HPMPC Administration                                                        Percent                                                              No. of   of                                       Body system                                                                              Preferred Term   Patients.sup.1                                                                         Patients                                 ______________________________________                                        Body as a Whole                                                                          asthenia         1        1.6                                                 allergic reaction (probenecid)                                                                 1        1.6                                                 infection (1 MAC & 1 PCP)                                                                      2        3.3                                      Cardiovascular                                                                           aneurysm         1        1.6                                      System                                                                        Hemic and  neutropenia (≦750 cells/mm.sup.3)                                                       7        11.5                                     Lymphatic System                                                                         neoplasm (lymphoma)                                                                            1        1.6                                      Special Senses                                                                           uveitis          1        1.6                                      Urogenital System                                                                        renal insufficiency (Cr ≧                                                               5        8.2                                                 2 mg/dL)                                                           ______________________________________                                         .sup.1 Of 61 total pahents who have received HPMPC.                      

In accordance with the results of preclinical animal studies, earlyclinical studies have identified nephrotoxicity as the major doselimiting toxicity of HPMPC. Similar dose limiting toxicity ultimatelymay be encountered with cHPMPC, but the animal studies described in theexamples below clearly demonstrate that the therapeutic window for thecyclic form is much wider than for HPMPC. However, at elevated doses ofcHPMPC, concomitant administration of agents with nephrotoxic potentialshould be avoided, if possible, and adherence to pre-cHPMPC doseexamination of urinalysis and serum chemistries will minimize thepotential for nephrotoxicity. Concomitant administration of hydrationand probenecid at cHPMPC doses approaching nephrotoxicity may benephroprotective, as is the case with HPMPC, although hydration isexpected to be of greatest value at such elevated cHPMPC doses.

Animals receiving massive doses of HPMPC also developed evidence of bonemarrow suppression and lymphoid depletion felt to be secondary to renalfailure and limited clearance of the drug. While neutropenia hasoccurred in patients receiving HPMPC in early clinical studies, this hasnot been dose-dependent and may be unrelated to HPMPC. It is unclearwhether similar effects will be encountered with cHPMPC, but theclinician would be advised to be alert for them.

Side effects associated with the administration of other antiviralmedications include sensory and motor neuropathy, central nervous systemdepression and agitation, headaches, nausea, vomiting, diarrhea,pancreatitis, hepatotoxicity, oral ulcers, cutaneous reactions, marrowsuppression, and nephrotoxicity. Any of these side effects are possiblewith cHPMPC.

Determination of Maximum Non-Cytotoxic Dosages of HSNA's; cHSNA Dosing

The term "maximum non-cytotoxic dose" (hereafter "MND") means themaximum molar quantitative amount of HSNA that can be administered tothe subject in question without inducing a toxic response that, in theopinion of the ordinary reasonable clinician, would necessitate areduction in dose of the HSNA or the withdrawal of the subject fromtreatment with the HSNA. The MND for a given subject will vary dependingon a number of factors, including the pre-existing condition of thepatient, (the MND will be lower if the patient already is demonstratinginjury to an organ for which the HSNA is cytotoxic), the nature of thecytotoxicity (potentially life-threatening cytotoxicities, e.g. fororgans such as kidney or liver, will lower the MND), the frequency ofadministration of the HSNA (giving the same dose of HSNA in disperseddoses as opposed to a bolus generally will lower the MND for the HSNAover a given period of time), the period that the subject has been onthe HSNA (longer periods of therapy on HSNA generally will lower the MNDfor subsequent dosings) and the presence or absence of concomitanttherapies that are expected to exacerbate or to ameliorate the expectedcytotoxicity. It is possible with minimal experimentation to determinethe MND for the ordinary subject, for example patients not bearing anyunusual pre-existing conditions and not requiring coadministration ofagents expected to exacerbate the HSNA cytotoxicity in question. ThisMND can be used to establish the initial dose for subsequent patients inthe same cohort. In any case, the practice of monitoring and optimizingtherapeutic dosing even in individual patients is a long standing andconventional practice, and it would not require any experimental effortoutside that which is ordinarily undertaken by the clinician.

Frequently encountered HSNA cytotoxicities include skin irritation (whenadministered topically), punctal stenosis (when administered byopthalmic modes of delivery, such as eyedrops) and nephrotoxicity bysystemic treatment as described above. cHSNAs are expected to exhibitsubstantially less of these cytotoxicities while still havingessentially the same antiviral activity, thereby permitting the molardose of cHSNA to exceed the MND of the corresponding HSNA. Since theantiviral activity of the cHSNA is essentially the same as the HSNA onan equimolar dose basis, administration of the cHSNA dose above the MNDof the HSNA will greatly increase therapeutic antiviral activity.

Nephrotoxicity is the dose-limiting toxicity for many HSNAs, and is thecurrent barrier to administration of larger doses of HPMPC by systemicroutes. Accordingly, the MND for systemically-administered HPMPC isequivalent to its maximum non-nephrotoxic dose.

The term "non-nephrotoxic dose" means a systemic dose administered by aroute, frequency and amount that fails to produce 2+ proteinuria asmeasured by urinalysis reagent strips. The "maximum non-nephrotoxicdose" means the greatest amount of HSNA by a given route and frequencythat can be administered to a subject without producing 2+ proteinuria.The patient being treated may be exposed to nephrotoxic agents or havepre-existing kidney damage, in which case the maximum non-nephrotoxicdose in these patients will be lower than in most patients. On thecontrary, supplemental therapies directed at amelioratingnephrotoxicity, e.g., administration of probenecid or adequatehydration, will cause the maximum non-nephrotoxic dose to be higher thanin patients not receiving such therapies. Accordingly, the range ofnon-nephrotoxic doses will vary somewhat from patient-to-patientdepending upon these and other factors known to the artisan. In general,one must take into account the condition of the patient, thedistribution of the dosage over time, the amount of time the patient hasbeen on drug, the administration route, the animal species beingtreated, the use of nephroprotective measures such as probenecid andhydration, and the concomitant administration of nephrotoxins. Themaximum non-nephrotoxic dose for HPMPC for humans in the ordinaryclinical setting generally is about 5 mg/kg weekly parenterally whenadministered with probenecid and hydration, or about 2 mg/kg weeklyparenterally without probenecid and hydration. The maximumnon-nephrotoxic doses of other HSNAs are determined by routinepreclinical or clinical experiments well within the ordinary skill inthe art as described above. A salient feature of this invention is thatsubstantially the same or greater antivirally efficacy of an HSNA can beachieved with the same dosage of cyclic analogue of the HSNA, but withmuch less toxicity, in particular nephrotoxicity. This means that theminimum antivirally active, non-nephrotoxic dose of the cHSNA will begreater than the maximum non-nephrotoxic dose of the HSNA on a molarbasis, all other therapeutic influences being essentially the same asnoted above.

Since cHPMPC is substantially less toxic, but similarly efficaciouscompared to HPMPC, one can employ substantially greater systemic molardoses of cHPMPC than the maximum non-nephrotoxic dose of HPMPC and stillnot induce nephrotoxicity in patients. In most embodiments, the typicalcHPMPC molar dosage will be greater than twice (on a molar basis) theHPMPC maximum non-nephrotoxic or non-cytotoxic dose, although it alsomay be 3, 4, 5, 6, 7, 8, 9, 10 times the maximum non-nephrotoxic ornon-cytotoxic dose. In most circumstances, a cHPMPC dosage of greaterthan about 10 mg/kg/week administered parenterally to humans will beantivirally effective and non-nephrotoxic. However, dosages of about 15,20, 25, 30, 35, 40, 45 or 50 mg/kg/week also may be suitable under thecircumstances. The greatest non-nephrotoxic dose of cHPMPC that can beused in humans is believed to be in the order of 50 mg/kg/week, but willvary based on the same parameters as the minimum dose, and may extend to100 mg/kg/week.

One also needs to take into account whether the HSNA or cHSNA is anintermediate that is converted in vivo into the free acid, i.e., whetherthe phosphonate hydroxyl group(s) are unsubstituted or are substitutedby A group(s). In general, the dosage of an intermediate form of cHSNAwill be higher than that of the free hydroxyl HSNA, taking into accountthe bioavailability of the intermediate by oral intake and its greatermolecular weight. The dosage of this invention for a given cHSNAintermediate is determined readily by assaying the proportion of freecHSNA generated in the plasma upon administration of the intermediate,generally by the oral route. The intermediate will be administered so asto emulate the desired cHSNA plasma concentration previously obtained byintravenous or other systemic administration routes. Analogous reasoningis applied to topical routes of administration, where the benchmark isthe tissue concentration at the topical site of delivery. In anillustrative example of the foregoing principles, if the maximumnon-nephrotoxic dose of the uncyclized HSNA by intravenousadministration is 1 mg/kg/day, then the intravenous dose of the cHSNAwill be greater than 1 mg/kg/day (and ordinarily greater than 2mg/kg/day). If the intermediate form of the cHSNA is 50% bioavailableupon oral administration and is 3 times the molecular weight of thecorresponding HSNA, then the oral dose of the cHSNA intermediate will begreater than about 6 mg/kg/day. The determination of bioavailability forsuch compounds is conventional and well within the ordinary skill in theart.

Alternatively, it will be within the skill of the ordinary artisan todetermine the MND for other HPMPB or HSNA compounds by simply elevatingthe dosages until evidence of nephrotoxicity (2+ proteinuria) or otherdose-limiting cytotoxicity is detected as described above. In general,initial dosages will be in the range of about 0.5 mg/kg to 10 mg/kgadministered 1, 2 or 7 times a week, and thereafter the amounts areincreased until toxicity is evident. Usually, only 1 or 2 animal speciesare studied, e.g., rats or guinea pigs, to arrive at non-cytotoxiccandidate doses for humans in accord with conventional practice.

HSNA Intermediates

Included within the scope of this invention are intermediates for thecyclized HSNA compounds. Such intermediates have structure (VIIIa).##STR15## wherein Z₂, A, Y, *, and B' are defined above.

Suitable A substituents are amidates or esters which may, but need notbe, hydrolyzable in vivo. Those which are not hydrolyzable in vivo areuseful as intermediates for in vitro hydrolytic conversion to the freeacids. Those that are hydrolyzable in vivo are useful as prodrugs. GroupA includes OD¹ wherein D¹ is a saccharide residue, a glyceride lipidresidue, unsubstituted C₁ -C₂₀ alkyl (but usually not C₁ -C₂ alkyl), C₂-C₂₀ alkenyl or alkynyl, C₂ -C₁₀ alkyoxyalkyl, C₄ -C₁₀ aryl, C₄ -C₁₀heteroaryl, C₅ -C₂₀ alkaryl, C₅ -C₂₀ alkoxyalkaryl, C₅ -C₂₀alkheteroaryl, --CH₂ C(O)NR₄, --CH₂ C(O)OR₄, --CH₂ OC(O)R₄,--CH(R₄)OC(O)R₄, --OC(R₄)HC(O)N(R₄)₂, --OC(R₄)HC(O)NH(R₄), --CH₂ C(R₄)₂CH₂ OH, or C₅ -C₂₀ alkoxyalkheteroaryl groups, or the same groups inwhich at least one (ordinarily 1-3) hydrogen atom is substituted withamino, hydroxyl, carboxyl, --OR₄, --COOR₄, --CON(R₄)₂, --CONH(R₄),--CONH₂, --NO₂, --CX₃, OCX₃, --CN, --N₃, or halo, where X is halo orhydrogen but at least one X is halo and R⁴ is C₁ -C₂₀ branched or normalalkyl, aryl or aralkyl which may be unsubstituted, or 1-3 hydrogen atomsof R⁴ are independently substituted with hydroxy, amino or halogen.

For the purposes herein, alkyl groups are branched or normal, arylgroups contain two fused rings, or are monocyclic, and heteroarylsubstituents contain 1 or 2 nitrogen atoms, an oxygen atom, an oxygenand a nitrogen atom, a sulfur atom, or a nitrogen and sulfur atom.Heteroatoms in aromatic rings with 2 ring heteroatoms ordinarily areseparated by at least one methylene group.

Suitable esters OD¹ are disclosed in U.S. Ser. No. 08/123,483 or EP481,214. These include D¹ =2,3-dihydro-6-hydroxyindene; sesamol;catechol monoester; C₃ -C₆ aryl (e.g. phenyl); C₃ -C₆ heteroaryl(including 2- and 3-pyrrolyl, 2- and 3-thienyl, 2- and 4-imidazolyl, 2-,4- and 5-oxazolyl, 3- and 4-isoxazolyl, 2-, 4- and 5-thiazolyl, 3-, 4-and 5-isothiazolyl, 3- and 4-pyrazolyl, 2-, 3- and 4-pyridinyl and 2-,4- and 5-pyrimidinyl); or such C₃ -C₆ aryl or heteroaryl groupssubstituted by halogen (1-5 atoms), C₁ -C₁₂ alkoxy (including methoxyand ethoxy) cyano, nitro, OH, C₁ -C₁₂ haloalkyl (1 to 6 halogen atoms),C₁ -C₁₂ alkyl (including methyl and ethyl), C₂ -C₁₂ alkenyl or C₂ -C₁₂alkynyl. Other D¹ groups include C₁ -C₄ alkyl- C₃ -C₆ -aryl (e.g.benzyl); C₁ -C₄ alkyl-C₃ -C₆ -heteroaryl (including --CH₂ -pyrrolyl,--CH₂ -thienyl, --CH₂ -imidazolyl, --CH₂ -oxazolyl, --CH₂ -isoxazolyl,--CH₂ -thiazolyl, --CH₂ -isothiazolyl, --CH₂ -pyrazolyl, --CH₂-pyridinyl and --CH₂ -pyrimidinyl); and such alkyl-C₃ -C₆ -aryl or-heteroaryl groups substituted at a hydrogen atom of the aryl orheteroaryl moiety by groups selected from halogen (1-5 atoms), C₁ -C₁₂alkoxy (including methoxy and ethoxy), cyano, nitro, OH, C₁ -C₁₂haloalkyl (1 to 6 halogen atoms), C₁ -C₁₂ alkyl (including methyl andethyl), C₂ -C₁₂ alkenyl or C₂ -C₁₂ alkynyl.

Exemplary D¹ groups include 2-, 3- and 4-alkoxyphenyl (C₁ -C₁₂ alkyl)including 2-, 3- and 4-methoxyphenyl, 2-, 3- and 4-ethoxyphenyl; 2-, 3-and 4-carboethoxyphenyl; 2- and 3-carboethoxy-4-hydroxyphenyl; 2- and3-ethoxy-4-hydroxyphenyl; 2- and 3-ethoxy-5-hydroxyphenyl; 2- and3-ethoxy-6-hydroxyphenyl; 2-, 3- and 4-O-acetylphenyl; 2-, 3- and4-dimethylaminophenyl; 2-, 3- and 4-methylmercaptophenyl; 2-, 3- and4-halophenyl (including 2-, 3- and 4-fluorophenyl and 2-, 3- and4-chlorophenyl); 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-dimethylphenyl;2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-biscarboxyethylphenyl; 2,3-, 2,4-,2,5-, 2,6-, 3,4- and 3,5-dimethoxyphenyl; 2,3-, 2,4-, 2,5-, 2,6-, 3,4-and 3,5-diethoxyphenyl); 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and3,5-dihalophenyl (including 2,4-difluorophenyl and 3,5-difluorophenyl);2-, 3- and 4-haloalkylphenyl (1 to 5 halogen atoms);4-trifluoromethylphenyl; 2-, 3- and 4-cyanophenyl; 2-, 3- and4-nitrophenyl; and 2-, 3- and 4-haloalkylbenzyl (1 to 5 halogen atoms),including 4 trifluoromethylbenzyl.

Acyloxymethyl esters (--OCH₂ OC(O)R³) or alkoxycarboxylmethyl esters(--OCH₂ C (O)OR³) are particularly useful. R³ is C₁ -C₁₅ (a) branched,normal or cycloalkyl, (b) unsubstituted monocyclic or polycyclic aryl or(c) either of the foregoing which are mono or disubstitutedindependently with --OR⁵, --R⁵, --NO₂, --CX₃, --OCX₃, or halo; R⁵ is C₁-C₅ branched or normal alkyl; and X is defined above.

Exemplary A groups also include residues of ∝-D-galactose, ∝-D-glucoseor ∝-D-fructose; enolpyruvate (HOOC-C(=CH₂)O--); glycerol; orD-∝,β-diglyceride. The fatty acids composing glyceride lipids generallyare naturally occurring saturated or unsaturated C₆ -C₂₆ fatty acidssuch as linoleic, lauric, myristic, palmitic, stearic, oleic,palmitoleic, linolenic and the like fatty acids.

Group A also is an amidate, e.g. --NHR⁵,--N(R⁵)₂, --NHC(=NH)N(CH₃)CH₂COOH, or an amino acid residue or peptide, wherein R⁵ is defined above.In the lattermost instance, an amino acid residue or peptide is linkedto the phosphorus atom through an epsilon or alpha amino group, therebyproducing a phosphoramidate bond. The amino acid residue is any moietycomprising at least one carboxyl and at least one amino residue linkedby at least one intervening carbon atom, typically a single (α) carbonatom, while peptides are polymers of two or more of such amino acids.

A variety of intervening structures located between the carboxyl andamino (amidate) groups are suitable. All that is necessary is that thegroup have sufficient conformation and length to be capable offacilitating acid catalysis of the phosphoroamidate bond and release ofthe phosphonate by the carboxyl group. The free carboxyl generally isproduced in vivo, e.g. by deesterification, deamidation or peptidolyticcleavage of a carboxyl ester or amide of the amino acid residue. Ingeneral, the intervening structure may be as simple as methylene (whenthe residue is glycyl) or substituted methylene (other α amino acids).The structure ordinarily contains up to about 5 carbon or heteroatoms inthe direct linkage between the carboxyl carbon and the amidate nitrogen,as for example in the case of intervening ethylene, propylene, butylene,or pentylene groups or their substituted analogs, such as for exampleoxyesters in which O replaces carbon. An example of such an interveningstructure would be --CH--O--CH(R⁷)(R⁶)--, where R⁶ and R⁷ are definedbelow. In general, fewer intervening atoms are employed when more rapidhydrolysis is desired, although it will be understood that largerstructures are suitable if they possess sufficient flexibility or arecapable of conformationally positioning the carboxyl group adjacent tothe amidate bond.

In general, the amino acid residues for use herein have the structureshown in structure (IX). ##STR16##

Ordinarily, n is 1 or 2, R⁶ is H and R⁷ is a moiety containing one ormore of the following groups: amino, carboxyl, amide, carboxyl ester,hydroxyl, C₆ -C₇ aryl, ether, n-, s- or t-alkyl (C₁ -C₆), guanidinyl,imidazolyl, indolyl, sulfhydryl, sulfoxide, and phosphoryl, or R⁷ istaken together with R⁹ to form C₃ -C₄ alkylene when n=1. Other R⁷ groupsinclude 1-guanidinoprop-3-yl, benzyl, 4-hydroxybenzyl, imidazol-4-yl,indol-3-yl, methoxyphenyl and ethoxyphenyl.

Ordinarily R⁶ is H and R⁷ is a side group or atom of a naturallyoccurring amino acid such as H, --CH₃, --CH(CH₃)₂, --CH₂ --CH(CH₃)₂,--CHCH₃ --CH₂ --CH₃, --CH₂ --C₆ H₅, --CH₂ CH₂ --S--CH₃, --CH₂ OH,--CH(OH)--CH₃, --CH₂ --SH, --CH₂ --C₆ H₄ OH, --CH₂ --CO--NH₂, --CH₂--CH₂ --CO--NH₂, --CH₂ --COOH, --CH₂ --CH₂ --COOH, --(CH₂)₄ --NH₂,--(CH₂)₃ -- R⁹ !, and --(CH₂)₃ --NH--C(NH₂)--NH₂. With respect to thecarboxyl-containing side chains of naturally occurring amino acids suchas glutamic and aspartic acid, it will be understood that if the C atomof the amino acid carboxyl group is linked by 5 or less atoms to thephosphoramide N atom then the carboxyl will be blocked, e.g. byesterification or amidation.

R⁸ is OD² or N(D²)₂ wherein D² is independently H or D¹ described above,but ordinarily is H, unsubstituted C₃ -C₉ alkyl, C₁ -C₅ alkyl-O--C₁ -C₅alkyl, phenyl, C₄ -C₆ cycloalkyl, or benzyl, or is C₃ -C₉ alkyl, C₁ -C₅alkyl-O--C₁ -C₅ alkyl, phenyl, C₄ -C₆ cycloalkyl, or benzyl substitutedwith OH, N(D²)₂, halogen, carboxyl, amide, or carboxyl ester (COOR⁴).

R⁹ is H or D², but usually is H.

When the amino acid residues contain one or more chiral centers, any ofthe D, L, meso, threo or erythro (as appropriate) racemates, or mixturesthereof are suitable. In general, if the HSNA intermediate is to behydrolyzed non-enzymatically in vivo, D isomers should be used. On theother hand, L isomers may be more versatile since they can besusceptible to both non-enzymatic as well as potential targetedenzymatic hydrolysis, and may be more efficiently transported by aminoacid or dipeptidyl transport systems in the gastrointestinal tract.

Examples of suitable amino acid residues include the following:

Glycyl;

Aminopolycarboxylic acids, e.g., aspartic acid, β-hydroxyaspartic acid,glutamic acid, β-hydroxyglutamic acid, β-methylaspartic acid,β-methylglutamic acid, β,β-dimethylaspartic acid, γ-hydroxyglutamicacid, β,γ-dihydroxyglutamic acid, β-phenylglutamic acid,γ-methyleneglutamic acid, 3-aminoadipic acid, 2-aminopimelic acid,2-aminosuberic acid and 2-aminosebacic acid residues;

Amino acid amides such as glutaminyl and asparaginyl;

Polyamino- or polybasic-monocarboxylic acids such as arginine, lysine,β-aminoalanine, γ-aminobutyrine, ornithine, citruline, homoarginine,homocitrulline, 5-hydroxy-2,6-diaminohexanoic acid (commonly,hydroxylysine, including allohydroxylysine) and diaminobutyric acidresidues;

Other basic amino acid residues such as histidinyl;

Diaminodicarboxylic acids such as α,α'-diaminosuccinic acid,α,α'-diaminoglutaric acid, α,α'-diaminoadipic acid, α,α'-diaminopimelicacid, α,α'-diamino-β-hydroxypimelic acid, α,α'-diaminosuberic acid,α,α'-diaminoazelaic acid, and α,α'-diaminosebacic acid residues;

Amino acids such as proline, 4- or 3-hydroxy-2-pyrrolidinecarboxylicacid (commonly, hydroxyproline, including allohydroxyproline),γ-methylproline, pipecolic acid, 5-hydroxypipecolic acid, --N( CH₂ !_(n)COOR⁴)₂, wherein n and R⁴ are as defined above, andazetidine-2-carboxylic acid residues;

A mono- or di-alkyl (typically C₁ -C₈ branched or normal) amino acidsuch as alanine, valine, leucine, allylglycine, butyrine, norvaline,norleucine, heptyline, α-methylserine, α-amino-α-methyl-γ-hydroxyvalericacid, α-amino-α-methyl-δ-hydroxyvaleric acid,α-amino-α-methyl-ε-hydroxycaproic acid, isovaline, α-methylglutamicacid, α-aminoisobutyric acid, α-aminodiethylacetic acid,α-aminodiisopropylacetic acid, α-aminodi-n-propylacetic acid,α-aminodiisobutylacetic acid, α-aminodi-n-butylacetic acid,α-aminoethylisopropylacetic acid, α-amino-n-propylacetic acid,α-aminodiisoamyacetic acid, α-methylaspartic acid, α-methylglutamicacid, 1-aminocyclopropane-1-carboxylic acid; isoleucine, alloisoleucine,tert-leucine, β-methyltryptophan and α-amino-β-ethyl-β-phenylpropionicacid residues; β-phenylserinyl;

Aliphatic α-amino-β-hydroxy acids such as serine, β-hydroxyleucine,β-hydroxynorleucine, β-hydroxynorvaline, and α-amino-β-hydroxystearicacid residues;

α-Amino, α-, γ-, δ- or ε-hydroxy acids such as homoserine,γ-hydroxynorvaline, δ-hydroxynorvaline and epsilon-hydroxynorleucineresidues; canavinyl and canalinyl; γ-hydroxyornithinyl;

2-hexosaminic acids such as D-glucosaminic acid or D-galactosaminic acidresidues;

α-Amino-β-thiols such as penicillamine, β-thiolnorvaline orβ-thiolbutyrine residues;

Other sulfur containing amino acid residues including cysteine;homocystine; β-phenylmethionine; methionine; S-allyl-L-cysteinesulfoxide; 2-thiolhistidine; cystathionine; and thiol ethers of cysteineor homocysteine;

Phenylalanine, tryptophan and ring-substituted α amino acids such as thephenyl- or cyclohexylamino acids α-aminophenylacetic acid,α-aminocyclohexylacetic acid and α-amino-β-cyclohexylpropionic acid;phenylalanine analogues and derivatives comprising aryl, lower alkyl,hydroxy, guanidino, oxyalkylether, nitro, sulfur or halo-substitutedphenyl (e.g., tyrosine, methyltyrosine and o-chloro-, p-chloro-,3,4-dicloro, o-, m- or p-methyl-, 2,4,6-trimethyl-, 2-ethoxy-5-nitro,2-hydroxy-5-nitro and p-nitrophenylalanine); furyl-, thienyl-, pyridyl-,pyrimidinyl-, purine or naphthylalanines; and tryptophan analogues andderivatives including kynurenine, 3-hydroxykynurenine,2-hydroxytryptophan and 4-carboxytryptophan residues;

α-Amino substituted amino acid residues including sarcosine(N-methylglycine), N-benzylglycine, N-methylalanine, N-benzylalanine,N-methyphenylalanine, N-benzylphenylalanine, N-methylvaline andN-benzylvaline; and

α-Hydroxy and substituted α-hydroxy amino acid residues includingserine, threonine, allothreonine, phosphoserine and phosphothreonineresidues.

Any amino acid is suitably employed as an A group provided that it iscapable of autocatalytically hydrolyzing the amidate bond. Thus, theymust contain, or must, upon being converted (hydrolyzed) in vivo, a freecarboxyl group. In general, the amino acids corresponding to theresidues employed in the compounds of this invention are naturallyoccurring and have no pharmacological activity. However, optimalpharmacokinetic activity (substantially complete autocatalytichydrolysis upon hydrolysis of the distal amide or ester bond) may beachieved by the use of non-naturally occurring amino acid residues.

Of particular interest are hydrophobic residues such as mono-or di-alkylor aryl amino acids, cycloalkylamino acids (proline) and the like. Thesehydrophobic residues, together with R⁸, contribute to cell permeabilityby increasing the partition coefficient of the nucleotide analogamidate. Typically, the residue will not contain a sulfhydryl orguanidino substituent.

If n1 is greater than 1, then Group A is a polypeptide radical,including dipeptides, short polypeptides of 3, 5 or 10 residues, orproteins having up to 100 or more residues. For the most part,dipeptides not containing aspartic or glutamic acid in the residueadjacent to the P atom will not autocatalytically hydrolyze the amidatebond and therefore the carboxyl groups (generally 1 or 2) in the distalresidue do not need to be esterified or amidated, i.e., R⁸ can be OH inthese circumstances. However, if such compounds are intended to be usedas precursors for the free phosphonate nucleotide analog in vivo, ratherthan as immunogens for example, the polypeptides ordinarily will containa peptidolytic enzyme cleavage site at the peptide bond linking thefirst residue and the next residue distal to the phosphorus atom. Suchcleavage sites are flanked by enzymatic recognition structures, e.g.particular residues recognized by a peptidolytic enzyme.

Peptidolytic enzymes are well known, and in particular includecarboxypeptidases. Carboxypeptidases digest polypeptides by removingC-terminal residues, and are specific in many instances for particularC-terminal sequences. Such enzymes and their substrate requirements ingeneral are well known. For example, a dipeptide having a given pair ofresidues and a free carboxyl terminus is covalently bonded through its∝-amino group to the phosphorus atom of the HSNA's of this invention. Itis expected that this peptide will be cleaved by the appropriatedipeptidase or protease, leaving the carboxyl of the proximal amino acidresidue to autocatalytically cleave the amidate bond.

Examples of dipeptidyl groups (designated by their single letter code)include AA, AR, AN, AD, AC, AE, AQ, AG, AH, AL, AL, AK, AM, AF, AP, AS,AT, AW, AY, AV, RA, RR, RN, RD, RC, RE, RQ, RG, RH, RI, RL, RK, RM, RF,RP, RS, RT, RW, RY, RV, NA, NR, NN, ND, NC, NE, NQ, NG, NH, NI, NL, NK,NM, NF, NP, NS, NT, NW, NY, NV, DA, DR, DN, DD, DC, DE, DQ, DG, DH, DI,DL, DK, DM, DF, DP, DS, DT, DW, DY, DV, CA, CR, CN, CD, CC, CE, CQ, CG,CH, CI, CL, CK, CM, CF, CP, CS, CT, CW, CY, CV, EA, ER, EN, ED, EC, EE,EQ, EG, EH, EI, EL, EK, EM, EF, EP, ES, ET, EW, EY, EV, QA, QR, QN, QD,QC, QE, QQ, QG, QH, QI, QL, QK, QM, QF, QP, QS, QT, QW, QY, QV, GA, GR,GN, GD, GC, GE, GQ, GG, GH, GI, GL, GK, GM, GF, GP, GS, GT, GW, GY, GV,HA, HR, HN, HD, HC, HE, HQ, HG, HH, HI, HL, HK, HM, HF, HP, HS, HT, HW,HY, HV, IA, IR, IN, ID, IC, IE, IQ, IG, IH, II, IL, IK, IM, IF, IP, IS,IT, IW, IY, IV, LA, LR, LN, LD, LC, LE, LQ, LG, LH, LI, LL, LK, LM, LF,LP, LS, LT, LW, LY, LV, KA, KR, KN, KD, KC, KE, KQ, KG, KH, KI, KL, KK,KM, KF, KP, KS, KT, KW, KY, KV, MA, MR, MN, MD, MC, ME, MQ, MG, MH, MI,ML, MK, MM, MF, MP, MS, MT, MW, MY, MV, FA, FR, FN, FD, FC, FE, FQ, FG,FH, FI, FL, FK, FM, FF, FP, FS, FT, FW, FY, FV, PA, PR, PN, PD, PC, PE,PQ, PG, PH, PI, PL, PK, PM, PF, PP, PS, PT, PW, PY, PV, SA, SR, SN, SD,SC, SE, SQ, SG, SH, SI, SL, SK, SM, SF, SP, SS, ST, SW, SY, SV, TA, TR,TN, TD, TC, TE, TQ, TG, TH, TI, TL, TK, TM, TF, TP, TS, TT, TW, TY, TV,WA, WR, WN, WD, WC, WE, WQ, WG, WH, WI, WL, WK, WM, WF, WP, WS, WT, WW,WY, WV, YA, YR, YN, YD, YC, YE, YQ, YG, YH, YI, YL, YK, YM, YF, YP, YS,YT, YW, YY, YV, VA, VR, VN, VD, VC, VE, VQ, VG, VH, VI, VL, VK, VM, VF,VP, VS, VT, VW, VY and VV, wherein the amidate bond is formed with thesecond residue.

Exemplary dipeptidyl A groups have the structure of formula (X) whereinR⁶ is H, R⁷ independently are the side chains of a naturally occurringamino acid, and R⁸ and R⁹ independently are as defined above. ##STR17##

Tripeptides also are useful. The A group sequence --X¹ ProX² (where X¹is any amino acid and X² is an amino acid, a carboxyl ester of Pro or H)will be cleaved by luminal carboxypeptidase to yield X¹ with a freecarboxyl, which in turn autocatalytically cleaves the amidate bond. X²usually will be benzyl ester. Thus, n1 usually is 1, 2 or 3, but mayrange up to 5, 10 or 100 or more residues.

If the amino acid residue has 2 or more amine groups, e.g., in the caseof lysinyl, arginyl or ornithinyl residues, then R⁷ represents the group-- C(R¹⁰)₂ !_(n2) N(R⁶)-- where n2 is 0 to 6, R¹⁰ is H, C₁ -C₂₀ alkyl,C₆ -C₂₀ aryl, C₇ -C₂₀ alkylaryl, C₇ -C₂₀ arylalkyl, C₁ -C₂₀ alkoxy, C₆-C₂₀ aryloxy or hydroxyl, and R⁶ is defined above. Such compounds willcontain a plurality of phosphonate moieties. For example when both theepsilon (ε)/delta (δ) and alpha (α) amino groups of lysine or ornithineare substituted with HSNA moieties the amidate contains and is believedto be capable of releasing two molecules of active drug, each expectedto emerge under different pharmacokinetics and therefore furthersustaining the drug release.

The compounds herein may or may not exclude the compounds of structure(VIIIa) disclosed in EP 481,214. However, as noted above, the compoundsherein include the cHPMB compounds (Formula V) of EP 481,214 wherein thestereochemistry at the phosphorous atom is (R) substantially free of (S)or (S) substantially free of (R).

Bases

Typically, B' is selected from structures (XI)-(XIV). ##STR18## whereinR¹⁵ is H, OH, F, Cl, Br, I, OR¹⁶, SH, SR¹⁶, NH₂, or NHR¹⁷ ;

R¹⁶ is C₁ -C₆ alkyl including CH₃, CH₂ CH₃, CH₂ CCH (2-propynyl), CH₂CHCH₂ (2-allyl), C₃ H₇ ;

R¹⁷ is C₁ -C₆ alkyl including CH₃, CH₂ CH₃, CH₂ CCH, CH₂ CHCH₂, C₃ H₇ ;

R¹⁸ is N, CF, CCl, CBr, CI, CR¹⁹ or CSR¹⁹, COR¹⁹ ;

R¹⁹ is H, C₁ -C₉ alkyl, C₂ -C₉ alkenyl, C₂ -C₉ alkynyl or C₇ -C₉aryl-alkyl unsubstituted or substituted by OH, O, N, F, Cl, Br or Iincluding CH₃, CH₂ CH₃, --CHCH₂, --CHCHBr, CH₂ CH₂ Cl, CH₂ CH₂ F, --CH₂CCH, --CH₂ CHCH₂, C₃ H₇, CH₂ OH, CH₂ OCH₃, CH₂ OC₂ H₅, --CH₂ OCCH, --CH₂OCH₂ CHCH₂, CH₂ C₃ H₇, CH₂ CH₂ OH, CH₂ CH₂ OCH₃, CH₂ CH₂ OC₂ H₅, --CH₂CH₂ OCCH, --CH₂ CH₂ OCH₂ CHCH₂, CH₂ CH₂ OC₃ H₇ ;

R²⁰ is N, CBr, CCl, CNH₂, C=N--NH₂, COH, CR¹⁹, C=S, or CH;

R²¹ is N, CH, CCN, CCF₃, CC≡CH or CC(O)NH₂ ;

R²² is H, OH, NH₂, SH, SCH₃, SCH₂ CH₃, SCH₂ CCH, SCH₂ CHCH₂, SC₃ H₇,NH(CH₃), N(CH₃)₂, NH(CH₂ CH₃), N(CH₂ CH₃)₂, NH(CH₂ CCH), NH(CH₂ CHCH₂),NH(C₃ H₇) or halogen (F, Cl, Br or I);

R²³ is H, OH, F, Cl, Br, I, SCH₃, SCH₂ CH₃, SCH₂ CCH, SCH₂ CHCH₂, SC₃H₇, OR¹⁶, NH₂, or NHR¹⁷ ; and

R²⁴ is O or S.

B' includes both protected and unprotected bases certain of which aredescribed above. Protecting groups for exocyclic amines and other labilegroups are known (Greene et al. "Protective Groups in OrganicSynthesis") and include N-benzyl, isobutyryl, 4,4-dimethoxytrityl (DMT)and the like. The selection of protecting group will be apparent to theordinary artisan and will depend upon the nature of the labile group andthe chemistry which the protecting group is expected to encounter, e.g.acidic, basic, oxidative, reductive or other conditions. Exemplaryprotected species are N⁴ -benzoylcytosine, N⁶ -benzoyladenine, N²-isobutyrylguanine and the like.

Exemplary bases include adenine, cytosine, guanine, hypoxanthine,inosine, thymine, uracil, xanthine, 2-aminopurine, 2,6-diaminopurine,2-amino-6-chloropurine, 8-aza derivatives of adenine, guanine,2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine,inosine and xanthine; 7-deaza-8-aza derivatives of adenine, guanine,2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine,inosine and xanthine; 1-deaza derivatives of adenine, guanine,2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine,inosine and xanthine; 7-deaza derivatives of adenine, guanine,2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine,inosine and xanthine; 3-deaza derivatives of adenine, guanine,2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine,inosine and xanthine; 6-azacytosine, 5-fluorocytosine, 5-chlorocytosine,5-iodocytosine, 5-bromocytosine, 5-methylcytosine, 5-bromovinyluracil,5-fluorouracil, 5-chlorouracil, 5-iodouracil, 5-bromouracil,5-trifluoromethyluracil, 5-methoxymethyluracil, 5-ethynyluracil,5-propynyluracil and the like.

Typical bases include adenine, 1-deazaadenine, 3-deazaadenine,7-deaza-8-azaadenine, 8-azaadenine, guanine, 2, 6-diaminopurine,2-aminopurine, cytosine, 6-azacytosine, 5-fluorocytosine,5-methylcytosine, 5-bromovinyluracil, 5-fluorouracil and5-trifluoromethyluracil.

In general, B' in the case of compounds of structure (VIIa) will havestructure (XI), while B' will be one of structures (VIII), (VIIII) or(XIV) in structures (IIa), (IIIa), (VIa), (Ia), (Va) and (IVa).

Methods of Manufacture of HSNAs

cHPMPC and the cyclic analogues of other HSNAs are prepared by a numberof methods from the free hydroxy phosphonic acid. These methods includetreatment with DCC in DMF (Ho et al., op cit.), reaction withVilsmeier's reagent (ClCH═N(CH₃)₂ Cl), or methods of phosphateactivation known per se. In one embodiment of this invention for thepreparation of cHSNA from the corresponding HSNA, the HSNA is (a)treated with ClCH═N(CH₃)₂ Cl to yield the phosphonylchloridate and (b)optionally the phosphonylchoridate is reacted with a nucleophile(preferably at low temperature, e.g. lower than about -20° C.) such asan alcohol or amine to produce one of the intermediates described above.In a further step the product of steps (a) or (b) are subject tohydrolysis or protonolysis (typically acid protonolysis) respectively toyield the cHSNA (treatment of the product of step (a)) or itsintermediate (treatment of the product of step (b)). Vilsmeier's reagentis advantageously produced in situ by combining SOCl₂, PCl₅, POCl₃,COCl₂ or the like with DMF. Advantageously, the product of step (a) isnot purified or separated from the reaction mixture before being reactedwith the nucleophile, a distinct economic advantage for this syntheticroute. The compounds of structure (Ia) and (Va) are readily made fromtheir uncyclized counterparts by the same methods, e.g. treatment withDCC in DMF.

Substituted and unsubstituted alkyl, aryl, heteroaryl, arylalkyl,heteroarylalkyl and other D¹ esters and amidates of cHSNA typically aremade by reacting the appropriate HSNA with SOCl₂ /DMF to yield theactivated phosphonylchloride (see Scheme 1), followed by treatment withthe corresponding nucleophile (e.g. alkoxide, phenolate, amine, etc.) toyield the protected intermediate formamidine which is subsequentlyhydrolyzed to the target compound. Alternatively, esters can also beprepared as depicted in Scheme 2. The N-,O- protected intermediatephosphonate diester is obtained from the three building blocks by knownmethods. The N- and O- protecting groups are subsequently removedfollowed by treatment of the phosphonate diester 3 with NaH leading tocyclization yielding target compound 4. A third method for the synthesisof cHSNA esters entails alkylation of the cHSNA using common alkylatingagents D¹ L (where L is a leaving group) such as alkyl halides,tosylates, diazoalkanes and the like (see Scheme 3). This method isparticularly useful for preparing acyloxyalkyl esters by treatment ofthe cHSNA with the corresponding acyloxyalkylhalide. In an exemplarymethod for the preparation of acyloxyalkyl esters of cHSNAs, shown inmore detail in Example 8, DCC and R⁴ C(O)OCH₂ Cl are reacted with thecHSNA; but in contradistinction with prior methods the stoichiometricproportion of DCC: R⁴ C(O)OCH₂ Cl, cHSNA is 1-2:1-2:1. Use of such lowproportions of reactants lessens side reactions with any exocyclic aminogroup of B' and thereby greatly improves yields.

Stereochemically pure compounds at the carbon chiral center are known orreadily prepared by known methods. The general method exemplified inExample 7 is useful in preparing a substantially pure phosphorus atomenantiomer of the intermediate compounds herein. If the general methodof Example 7 is not suitable to yield the desired enantiomer then theracemate is prepared and the enantiomers separated by conventionalmethods, e.g. chromatography. In general, this separation will besimplified if the carbon chiral center is a pure enantiomer obtained byjudicious selection of appropriate starting materials, whereby only thephosphorus atom is racemic.

Each of the following schemes exemplify cytosine as the base. However,any B' is employed in place of cytosine, provided that any exocyclic oxoor amino groups are protected as required. Also, step 3 of scheme 1obviously will be omitted when B' contains no exocyclic amine. ##STR19##

Salts

The compounds of this invention optionally are supplied as salts. Thosesalts which are pharmaceutically acceptable are of particular interestsince they are useful in administering the foregoing compounds formedical purposes. Salts which are not pharmaceutically acceptable areuseful in manufacturing processes, for isolation and purificationpurposes, and in some instances, for use in separating stereoisomericforms of the compounds of this invention. The latter is particularlytrue of amine salts prepared from optically active amines.

Pharmaceutically acceptable metal and amine salts are useful herein andinclude salts which are stable under ambient conditions and whichcontain nontoxic cations. Suitable metal salts include the sodium,potassium, calcium, barium, zinc, and aluminium salts. The sodium andpotassium salts are preferred. Suitable amine salts are prepared fromamines which have sufficient basicity to form a stable salt, andpreferably include those amines which are frequently used in medicinalchemistry because of their low toxicity and acceptability for medicaluse. These include ammonium and the trialkylamines such astriethylamine, and others including procaine, dibenzylamine,N-benzyl-beta-phenethylamine, ephenamine, N,N'-dibenzylethylenediamine,dehydroabietylamine, N-ethylpiperidine, benzylamine, basic amino acids,e.g. lysine and arginine, and dicyclohexylamine.

Acid addition salts are formed with the compounds of the invention inwhich a basic function such as an amino, alkylamino, or dialkylaminogroup is present as a substituent on B'. The pharmaceuticallyacceptable, i.e., nontoxic, acid addition salts are preferred. They arechosen optimally to be compatible with the customary pharmaceuticalvehicles and adapted for oral or parenteral administration. Somesuitable acids for use in the preparation of such salts are hydrochloricacid, hydrobromic acid, phosphoric acid, sulfuric acid, various organiccarboxylic and sulfonic acids, such as acetic acid, citric acid,propionic acid, succinic acid, benzoic acid, tartaric acid, fumaricacid, mandelic acid, ascorbic acid, malic acid, methanesulfonic acid,toluenesulfonic acid, fatty acids, and others.

Pharmaceutical Formulations

Compounds of the invention and their pharmaceutically, i.e.physiologically, acceptable salts (hereafter collectively referred to asthe active ingredients) may be administered by any route appropriate tothe condition to be treated, suitable routes including oral, rectal,nasal, topical (including ocular, buccal and sublingual), vaginal andparenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural). It will be appreciated that thepreferred route may vary with for example the condition of therecipient.

While it is possible for the active ingredients to be administered aloneit is preferably to present them as pharmaceutical formulations. Theformulations, both for veterinary and for human use, of the presentinvention comprise at least one active ingredient, as above defined,together with one or more acceptable carriers therefor and optionallyother therapeutic ingredients. The carrier(s) must be "acceptable" inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the patient.

The formulations include those suitable for topical or systemicadministration, including oral, rectal, nasal, buccal, sublingual,vaginal or parenteral (including subcutaneous, intramuscular,intravenous, intradermal, intrathecal and epidural) administration. Theformulations are in unit dosage form and are prepared by any of themethods well known in the art of pharmacy. Such methods include the stepof bringing into association the active ingredient with the carrierwhich constitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as solution or a suspension in an aqueous liquid ora non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

For infections of the eye or other external tissues, e.g. mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w (including active ingredient(s) in a range between 0.1%and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc),preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredients may be formulated in a cream with an oil-in-watercream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. The topical formulations maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas. Examplesof such dermal penetration enhancers include dimethyl sulphoxide andrelated analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theemulsifying wax, and the wax together with the oil and fat make up theemulsifying ointment base which forms the oily dispersed phase of thecream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween® 60, Span® 80, cetostearylalcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate andsodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. Thus the cream shouldpreferably be a non-greasy, non-staining and washable product withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is suitably present in suchformulations in a concentration of 0.01 to 20%, in some embodiments 0.1to 10%, and in others about 1.0% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for nasal or inhalational administration whereinthe carrier is a solid include a powder having a particle size forexample in the range 1 to 500 microns (including particle sizes in arange between 20 and 500 microns in increments of 5 microns such as 30microns, 35 microns, etc). Suitable formulations wherein the carrier isa liquid, for administration as for example a nasal spray or as nasaldrops, include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol administration may be preparedaccording to conventional methods and may be delivered with othertherapeutic agents. Inhalational therapy is readily administered bymetered dose inhalers.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration are sterile andinclude aqueous and non-aqueous injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials with elastomeric stoppers, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed. Preferred unit dosage formulations are those containing adaily dose or unit daily sub-dose, as recited above, or an appropriatefraction thereof, of an active ingredient.

In addition to the ingredients particularly mentioned above theformulations of this invention may include other agents conventional inthe art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavoringagents.

The present invention further provides veterinary compositionscomprising at least one active ingredient as above defined together witha veterinary carrier therefor.

Veterinary carriers are materials useful for the purpose ofadministering the composition to cats, dogs, horses, rabbits and otheranimals and may be solid, liquid or gaseous materials which areotherwise inert or acceptable in the veterinary art and are compatiblewith the active ingredient. These veterinary compositions may beadministered orally, parenterally or by any other desired route.

Compounds of the invention can be used to provide controlled releasepharmaceutical formulations containing a matrix or absorbent materialand as active ingredient one or more compounds of the invention in whichthe release of the active ingredient can be controlled and regulated toallow less frequent dosing or to improve the pharmacokinetic or toxicityprofile of the compound. Controlled release formulations adapted fororal administration in which discrete units comprising one or morecompounds of the invention can be prepared according to conventionalmethods.

Anti-Infective Activity

The invention compounds are used in the treatment or prophylaxis ofvarious microbial infections particularly human bacterial, humanparasitic protozoan or human viral infections caused by microbialspecies including Plasmodium, Pneumocystis, herpesviruses (CMV, HSV 1,HSV 2, VZV, and the like), retroviruses, hepadnaviruses, (e.g. HBV),papillomavirus, hantavirus, adenoviruses and the like. The controlledrelease formulations can be used to treat HIV or HIV-relatedopportunistic infections and related conditions such as tuberculosis,malaria, pneumocystis pneumonia, and CMV retinitis. Other retroviralinfections that may be treated with the compounds according to theinvention include Human T-cell Lymphotropic virus (HTLV)-I and IV andHIV-2 infections, MSV, RSV, SIV, FIV, MuLV, and other retroviralinfections of rodents and other animals.

The following examples are intended to illustrate the invention and arenot to be construed as limiting the scope of the claims. All citationsherein are expressly incorporated by reference.

EXAMPLE 1

5 Day Repeat Dose Toxicity Study of HPMPC, cHPMPC and EtHPMPCAdministered Intravenously to Rats

A histopathological evaluation was performed on the kidneys from thirtymale and thirty female Sprague-Dawley rats. The purpose was to evaluatethe potential nephrotoxicity in rats following repeated intravenousonce-a-day administration of either HPMPC, cHPMPC or EtHPMPC(monoethylphosphonate ester of HPMPC) for five consecutive treatmentdays and a 10 day post dosing observation period. Comparison was made torats receiving vehicle (sterile 0.9% Saline for injection, USP). Waterwas available ad lib to the rats. The rats were divided into thefollowing six groups:

    ______________________________________                                                           Dose Level  Animals on Study                               Group    Test Article                                                                            (mg/kg/day) Male Female                                    ______________________________________                                        1        Vehicle.sup.a                                                                           0.0         5    5                                         2        HPMPC     100.0       5    5                                         3        cHPMPC    100.0       5    5                                         4        cHPMPC    250.0       5    5                                         5        EtHPMPC   100.0       5    5                                         6        EtHPMPC   250.0       5    5                                         ______________________________________                                         .sup.a Sterile 0.9% Saline for injection, USP.                           

Complete necropsies were performed on all of the animals and selectedtissues were fixed and preserved in 10% neutral buffered formalin.Paraffin embedded, hematoxylin and eosin stained section of the kidneyswere prepared by Experimental Pathology Laboratories, Inc. and evaluatedfrom all of the animals.

Microscopic findings for each tissue examined from each animal arelisted in the Histopathology Incidence Tables 3 and 4 below.Inflammatory, degenerative and hyperplastic changes were graded from oneto five depending upon severity; nongradable changes were designated aspresent (P) in the Histopathology Incidence Tables. All lesions aresummarized by treatment group and sex in the Summary Incidence Tablestogether with the total number of animals in each group for which thetissues were examined. The descriptions of the gross findings on thesetables were transcribed from the Individual Animal Necropsy Sheets.Gross changes observed at the time of tissue processing are indicated as"(noted at gross trimming)".

Treatment related changes were present in the kidneys of the male andfemale rats receiving 100.0 mg/kg/day of HPMPC but not in the kidneys ofthe male or female rats receiving 100.0 and 250.0 mg/kg/day of cHPMPC or100.0 and 250.0 mg/kg/day of EtHPMPC. These kidney changes in the ratsreceiving 100.0 mg/kg/day of HPMPC were characterized by a minimal tomoderately-severe tubular depletion and degeneration accompanied by aminimal to moderate tubular cytomegaly and tubular karyomegaly andminimal to moderately-severe tubular regeneration of the outer corticaltubules. In the more severely affected kidneys there was a loss of renaltubules in the outer cortical region. There were little or noinflammatory changes in these kidneys. The kidney changes were moresevere in the males than in the females.

                                      TABLE 3                                     __________________________________________________________________________    SUMMARY INCIDENCE                                                             Terminal Sacrifice                                                            Male Rat                                                                                   GROUP                                                                             GROUP                                                                             GROUP                                                                             GROUP                                                                             GROUP                                                                             GROUP                                                     1   2   3   4   5   6                                            __________________________________________________________________________    KIDNEY (NO. EXAMINED)                                                                      (5) (5) (5) (5) (5) (5)                                          Congestion   4       4   2   2   5                                            Cyst(s) Cortical                                                                           1       1   1   1                                                Mononuclear Cells                                                                              5   1           1                                            Nephritis, Nonsuppurative,                                                                 3       2   4   1   1                                            Multifocal                                                                    Nephritis, Subacute,                                                                           3                                                            Multifocal                                                                    Pelvic Dilatation. Bilaterat                                                               1       1                                                        Pelvic Dilatation, Unilateral                                                              1       1                                                        Tubular Basophilia                                                                             2                                                            Tubular Cytomegaly                                                                             5                                                            Tubular Depletion/                                                            Degeneration     5                                                            Tubular Dilatation                                                                         1   5       1   1   1                                            Tubular Karyomegaly                                                                            5                                                            Tubular Mineralization                                                        Tubular Regeneration                                                                       3   5   4   5   3   1                                            __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    SUMMARY INCIDENCE                                                             Terminal Sacrifice                                                            Female Rat                                                                                 GROUP                                                                             GROUP                                                                             GROUP                                                                             GROUP                                                                             GROUP                                                                             GROUP                                                     1   2   3   4   5   6                                            __________________________________________________________________________    KIDNEY (NO. EXAMINED)                                                                      (5) (5) (5) (5) (5) (5)                                          Congestion   3   2   4   3   4   4                                            Cyst(s). Cortical    1   1   1                                                Mononuclear Cells                                                                              5   1   1                                                    Nephritis. Nonsuppurative,                                                                 1   1   2   2   1   1                                            Multifocal                                                                    Nephritis. Subacute.                                                          Multifocal                                                                    Pelvic Dilatation, Bilateral                                                  Pelvic Dilatation, Unilateral                                                              1           1   1                                                Tubular Basophilia                                                                             5                                                            Tubulat Cytomegaly                                                                             4                                                            Tubular Depletion/                                                                             4                                                            Degeneration                                                                  Tubular Dilatation                                                                             3   1                                                        Tubular Karyomegaly                                                                            4                                                            Tubular Mineralization                                                                     3   2   1           2                                            Tubular Regeneration                                                                       3   5   2   2   2   1                                            __________________________________________________________________________

Incidental changes in the kidneys of a few control and treated rats hadchanges compatible with the early microscopic changes of old ratnephropathy. These microscopic changes consisted of one or more of thefollowing: multifocal nonsuppurative nephritis, tubular dilatationand/or tubular regeneration. Additional incidental kidney lesions wereunilateral or bilateral pelvic dilatation.

A few microscopic findings were observed in the kidneys of individualrats and occurred as incidental findings or in such small numbers as tohave no apparent relationship to the test material. These changes wereof the usual number, type and frequency observed in this strain andtheir presence did not interfere with the evaluation of the testmaterial used in this study.

EXAMPLE 2

14-Day Intravenous Toxicity Study of cHPMPC in Rats

A histopathological evaluation was performed on selected tissues fromtwenty male and twenty female Sprague-Dawley rats. The purpose was toevaluate the potential systemic toxicity of cHPMPC in rats followingrepeated intravenous administration (once-a-day) over a 14 day treatmentperiod. Comparison was made to rats receiving vehicle (0.9% Saline).Water was available ad lib to the rats. The rats were divided into thefollowing four groups:

    ______________________________________                                                 Test     Dose Level   Animal Study                                   Group    Article  (mg/kg/day)  Male Female                                    ______________________________________                                        I        Vehicle.sup.a                                                                          0.0          5    5                                         II       cHPMPC   10.0         5    5                                         III      cHPMPC   40.0         5    5                                         IV       cHPMPC   150.0        5    5                                         ______________________________________                                         .sup.a 0.9% Saline.                                                      

Complete necropsies were performed on all of the animals and selectedtissues were fixed and preserved in 10% neutral buffered formalin.Paraffin embedded, hematoxylin and eosin stained sections of thefollowing tissues were prepared by Experimental Pathology Laboratories,Inc. from the rats in groups I and IV: esophagus, intestine, large(cecum, colon and rectum), intestine, small (duodenum, jejunum andileum), liver, lymph nodes (mesenteric), ovaries (female), spleen,stomach, testes (male), urinary bladder and injection site. The kidneysand liver were processed and evaluated from the rats in Groups II andIII.

Treatment related changes were present in the kidneys of the male andfemale rats receiving 40 and 150 mg/kg/day of cHPMPC but not in thekidneys of the male or female rats receiving 10 mg/kg/day of cHPMPC. Thekidney changes were more severe in the males than in the females. Notreatment related lesions were present in the esophagus, largeintestine, liver, mesenteric lymph nodes, ovaries, spleen, smallintestine, stomach, testes, urinary bladder or injection site in themale and female rats receiving 150 mg/kg/day of cHPMPC for 14 days. Thetoxic dose in this 14-day study was lower than in the 5-day study due tothe length of therapy.

The kidney changes in the rats receiving 150 mg/kg/day of cHPMPC werecharacterized by a minimal to moderately-severe tubular depletion anddegeneration accompanied by a minimal to moderate tubular cytomegaly andtubular karyomegaly and an increased incidence and/or severity oftubular regeneration and/or tubular dilatation. In the more severelyaffected kidneys there was a loss of renal tubules in the outer corticalregion. Little or no inflammatory changes were present in these kidneys.In the rats receiving 40 mg/kg/day of cHPMPC, there was a minimaltubular cytomegaly and tubular karyomegaly in two males and an increasedincidence of tubular regeneration in the females. These changes in thekidneys are summarized in Table 5.

                  TABLE 5                                                         ______________________________________                                        TREATMENT      Vehicle          GS930                                         GROUP          I        II      III    IV                                     DOSE (mg/kg/day)                                                                             0        10.0    40.0   150.0                                  NUMBER EXAMINED/SEX                                                                          5M    5F     5M  5F  5M  5F   5M  5F                           Tubular Depletion/                                                                           (0)   (0)    (0) (0) (0) (0)  (5) (5)                          Degeneration (No.)                                                            Minimal                                          5                            Slight                                       2                                Moderate                                     2                                Moderately severe                            1                                Tubular Cytomegaly (No.)                                                                     (0)   (0)    (0) (0) (2) (0)  (5) (5)                          Minimal                             2            5                            Slight                                       2                                Moderate                                     2                                Moderately severe                            1                                Tubular Karyomegaly (No.)                                                                    (0)   (0)    (0) (0) (2) (0)  (5) (5)                          Minimal                             2            5                            Slight                                       2                                Moderate                                     2                                Moderately severe                            1                                Tubular Dilatation (No.)                                                                     (1)   (1)    (1) (0) (1) (0)  (5) (3)                          Minimal        1     1                       2   3                            Slight                      1       1        1                                Moderate                                     1                                Moderately severe                            1                                Tubular Regeneration (No.)                                                                   (2)   (3)    (2) (1) (1) (5)  (5) (4)                          Minimal        1     2      1   1   1   5    4   3                            Slight         1     1      1                    1                            Moderate                                     1                                ______________________________________                                    

Incidental changes were present in the kidneys of a few control andcHPMPC treated rats. These microscopic changes consisted of one or moreof the following: congestion, cortical cysts, mononuclear cells,multifocal nonsuppurative nephritis, tubular dilatation and/orregeneration, tubular mineralization and unilateral or bilateral pelvicdilatation.

Incidental changes were present in the livers of the control and cHPMPCtreated rats. The more common of these were congestion, multifocalhepatocellular degeneration (single cell necrosis), multifocalnonsuppurative hepatitis, pericholangeal mononuclear cells andmultifocal hepatocellular vacuolation. Although no treatment relatedchanges were present in the livers of the male and female rats receiving10, 40 and 150 mg/kg/day of cHPMPC the severity of the incidentalchanges was slightly higher in the rats receiving cHPMPC.

A variety of microscopic findings were observed in individual rats andoccurred as incidental findings or in such small numbers as to have noapparent relationship to the test material. The degree of hematopoiesisin the spleens varied in the individual rats within groups and betweensexes but was considered to be within normal limits for the age andstrain of rats. The testes of the control males and the males receiving150 mg/kg/day of cHPMPC had normal spermatogenesis. The ovaries of thecontrol females and females receiving 150 mg/kg/day of cHPMPC had normalfollicular activity. A few additional findings were observed inindividual rats and occurred as incidental findings or in such smallnumbers as to have no apparent relationship to the test material. Thesewere of the usual type and incidence seen in rats.

The incidental findings occurred in both treated and control rats atessentially comparable incidence and severity and were of the usual typeand incidence commonly seen in rats. The presence of the incidentallesions did not interfere in the evaluation of the intravenousadministration of cHPMPC, as used in this study.

These results with cHPMPC at 40 mg/kg/day compare quite favorably with,and are comparable to, the results with Sprague-Dawley rats using only 3mg/kg/day of HPMPC by iv injection for 14 consecutive days (male ratsonly, 5 per group) as shown below in Tables 6a and 6b.

                  TABLE 6a                                                        ______________________________________                                                   HPMPC (mg/kg/day)                                                             Control                                                                       (Saline)                                                                              0.3     1.0       3.0                                                 Group 5 Group 6 Group 7   Group 8                                  ______________________________________                                        Kidney       (5)       (5)     (5)     (5)                                    Focal interstitial nephritis                                                               5         5       5       2                                      minimal      5         5       5       2                                      Inflammatory infiltrate                                                                    0         0       0       4                                      minimal      0         0       0       3                                      slight       0         0       0       1                                      Tubular nephrosis                                                                          0         0       0       5                                      slight       0         0       0       2                                      moderate     0         0       0       2                                      marked       0         0       0       1                                      ______________________________________                                    

                  TABLE 6b                                                        ______________________________________                                                        HPMPC  HPMPC                                                                  10 mg/kg                                                                             50 mg/kg                                                               Group 1                                                                              Group 2                                                ______________________________________                                        Kidney            (5)      (5)                                                Dilated tubule(s) 0        0                                                  Focal interstitial nephritis                                                                    0        0                                                  Inflammatory infiltrate                                                                         5        5                                                  minimal           0        2                                                  slight            5        3                                                  Tubular nephrosis 5        5                                                  marked            3        0                                                  severe            2        5                                                  ______________________________________                                    

EXAMPLE 3

Efficacy of HPMPC, cHPMPC and EtHPMPC Against HSV-2 EncephalitisInfection in Mice

In a preliminary study the antiviral activities of HPMPC, cHPMPC andEtHPMPC against herpes simplex virus type 2 (HSV-2) encephalitisinfection in mice were evaluated. In that study the efficacies of thethree compounds were very similar using doses of 3,1, 0.3 and 0.1mg/kg/day. However, the overall infection was mild since only 50% ofplacebo-treated mice died. Because of the low mortality rate, this castsome doubt as to the validity of the results with respect to therelative potencies of these compounds. For this reason the present studywas conducted whereby the virus challenge dose was adjusted to cause amore severe infection. This time it appeared that two of the compounds,HPMPC and cHPMPC, were similar in protective activity, and EtHPMPC waspoorly active if not inactive.

Compounds: HPMPC, cHPMPC, and EtHPMPC were supplied in dry powder form.They were made up in sterile saline for intraperitoneal (i.p.)administration and stored frozen between treatments. Sterile salineserved as the placebo control.

Infection: Swiss Webster female mice (Simonsen Labs, Gilroy Calif.)weighing approximately 17 grams each at the start of the experiment wereinfected i.p. with HSV-2 (MS strain) at 2×10⁵ plaque forming units (PFU)per mouse. This differs from the preliminary experiment where the miceweighed approximately 20 grams each and received 1×10⁵ PFU of virus.This slight adjustment in methodology was important to improve thepercentage of mortality in placebo-treated mice.

Treatment: Three hours after virus inoculation, i.p. treatments withcompounds and placebo were begun. Treatments were once daily for 5 days.

Parameters used to evaluate the infection: These included death and meanday to death determinations. Deaths were recorded daily for 21 days. Themean day of death calculation took into account only mice that died.Statistical interpretations of survival (Fisher Exact Test) and mean dayto death (Mann Whitney U-Test) were made by two-tailed analyses.

Table 7 shows the results of the experiment, indicating that HPMPC wassignificantly effective in reducing mortality at 1 and 3 mg/kg/day, withlower doses being ineffective. Likewise, cHPMPC caused significantreductions in mortality at 1 and 3 mg/kg/day. EtHPMPC proved to beinactive at the doses tested. Only HPMPC at 0.3 mg/kg/day caused asignificant increase in the mean day to death of mice that died,although doses of 1 and 3 mg cHPMPC/kg/day appeared to extend the lifespan.

                  TABLE 7                                                         ______________________________________                                        Effect of Three Antiviral Substances on HSV-2 (MS Strain)                     Included Encephalitis in Mice                                                           Dose.sup.a  Survivors/  Mean Day                                    Compound  (mg/kg/day) Total (%)   to Death                                    ______________________________________                                        HPMPC     3           10/10 (100)**                                                                             >21                                         HPMPC     1            9/10 (90)**                                                                               9.0 ± 0.0                               HPMPC     0.3          3/10 (30)  10.9 ± 2.3*                              HPMPC     0.1          3/10 (30)  10.9 ± 4.8                               cHPMPC    3            8/10 (80)**                                                                              14.0 ± 4.2.sup.b                         cHPMPC    1            8/10 (80)**                                                                              18.0 ± 28.sup.b                          cHPMPC    0.3          3/10 (30)  11.0 ± 3.1                               cHPMPC    0.1          0/10 (0)    9.8 ± 2.3                               EtHPMPC   3            3/10 (30)  10.0 ± 2.0                               EtHPMPC   1            1/10 (10)  10.0 ± 2.1                               EtHPMPC   0.3          1/10 (10)   8.8 ± 1.6                               EtHPMPC   0.1          2/10 (20)   8.5 ± 1.4                               Placebo   --           3/30 (10)   8.8 ± 1.3                               ______________________________________                                         .sup.a Intraperitoneal treatments were once daily for 5 days starting 3       hours after virus challenge.                                                  .sup.b Although these values appear to be statistically significant, ther     are too few data points for analysis.                                         *P < 0.05, **P < 0.001.                                                  

The foregoing examples unexpectedly demonstrate that cHPMPC is up toabout 13-fold less toxic than its uncyclized congener, HPMPC, but isquite similar in its antiviral activity. We expect that similar effectswill be observed for other HSNAs as well.

EXAMPLE 4

cHPMPC was synthesized by adding to a stirred suspension of HPMPC (100g, 0.358 mol) in DMF (2L) N, N'-dicyclohexyl-4-morpholine-carboxamidine(115 g, 0.393 mol). The reaction mixture was stirred for 12 hours atroom temperature. This solution was added slowly to a hot pyridinesolution (5 L, 60° C.) of DCC (185 g, 0.895 mol) through an additionfunnel. The reaction mixture was stirred at 100° C. for 16 hours, cooledto room temperature and the solvents were removed under reducedpressure. The crude mixture was washed with diethyl ether (3 L),dissolved in water (2 L) and washed with CH₂ Cl₂ (5×1 L). The aqueouslayer was concentrated to 1 L volume and acidified to pH-3.5. Uponcooling cyclic-HPMPC crystallized (89 g, ˜95% pure). The cHPMPC wasrecrystallized by dissolving in water at pH8 (with 1N NaOH) followed byacidification to pH 3.5 (with 1N HCl):

CHN Analysis: cHPMPC monohydrate, monosodium salt. C₈ H₁₁ N₃ O₅ PNa.H₂O: theory: C31.90 H4.69 N13.96%; found: C32.39 H4.91 N13.95%; 32P-NMR:9.35(s) (reference H₃ PO₄); ¹ H-NMR: 3.70-4.27 (m, 7H), 4.80 (s,HDO),6.15 (d, J=7.8, 1H), 7.83 (d, J =7.8, 1H). ¹³ C NMR (75 MHz, D₂ O) d,169.4 s (4-C), 161.0 s (2-C), 150.2 s (6-C), 98.21 s (5-C), 76.86 d(JP,C=3.6 Hz, 2'--CH₂), 72.44 d (JP,C=6.3 Hz, 3'--CH₂), 67.88 d(JP,C=143.0 Hz, P-CH₂), 51.90 s (1'-C).

EXAMPLE 5

cHPMPU was synthesized by adding thionyl chloride (60 mL, 0.812 mmol,2.02 eq) dropwise to a suspension of disodium HPMPU (131 mg, 0.404 mol)in N,N-dimethylformamide (1.25 mL) at ambient temperature. The resultinglight-yellow solution was stirred for 20 min at ambient temperature andthen concentrated to dryness (in vacuo, 45° C.). H₂ O (2 mL) was addedand the resulting solution was concentrated to dryness. Methanol (4 mL)was added and the resulting solution was concentrated to dryness toafford the crude product as a light-yellow solid. Purification by silicaflash chromatography (mobile phase: 30% methanol: 70% CH₂ Cl₂ gradientto 50% methanol: 50% CH₂ Cl₂) afforded pure cHPMPU in 69% yield as awhite amorphous solid. ¹ H NMR (300 MHz, D₂ O) d 7.62 d (1H, J=7.1 Hz,CH═CH), 5.82 d (1H, J=7.8 Hz, CH═CH), 4.30-3.71 m (7H, CH₂ CH(OCH₂ P)CH₂OH), NH and OH not observed in D₂ O. ¹³ C NMR (75 MHz, D₂ O) d, 169.6 s(4-C), 155.1 s (2-C), 150.4 s (6-C), 104.2 s (5-C), 76.71 d (JP,C=3.6Hz, 2'--CH₂), 72.30 d (JP,C=6.2 Hz, 3'--CH₂), 67.90 d (JP,C=142.0 Hz,P--CH₂), 50.71 s (1'-C). ³¹ P NMR (121 MHz, D₂ O) d 9.23 s.

EXAMPLE 6 ##STR20##

To a stirred solution of diethyl HPMPC (1.1 g) in DMF, NaH (115 mg) wasadded. After 15 min, the reaction mixture was quenched with acetic acid(1 eq). The solvents were removed under reduced pressure. The crudemixture was dissolved in CH₂ Cl₂ and water. The organic layer was washedwith NaCl solution and the crude material obtained was purified on asilica gel column (elution with 5%-10% MeOH in CH₂ Cl₂) to get cyclicethyl HPMPC (950 mg) as a diastereomeric mixture (approximately 70%).

EXAMPLE 7 ##STR21##

To a stirred suspension of HPMPC (2.79 g) in DMF, thionylchloride (2.1mL) was added dropwise under anhydrous conditions and the mixture wasstirred for 1 hr. In another flask, sodium aryloxide (using theappropriate aryl substituent) was made using the corresponding phenol(8.9 g) and NaH (1.8 g) in 1:1 DMF/THF (50 mL). This solution was cooledto -78° C. and the chloridate solution was added dropwise underanhydrous conditions. After 2 hrs, the reaction mixture was quenchedwith acetic acid (5 eq) and the solvents were evaporated under vacuum.The crude mixture was partitioned between water and CH₂ Cl₂. The organiclayer was concentrated and the residue was purified on a silica gelcolumn (elution with 5%-10% MeOH in CH₂ Cl₂) to get the cyclic arylcompound as a single diastereomer in approximately 60% yield. Thismethod is suitable for all substituted or unsubstituted D' groups,especially aryl, subject of course to conventional protection of labilegroups other than amino for which reaction is undesired (amino isprotected by reaction with DMF and deprotected with acetic acid andalkanol treatment). This method offers the advantages of producingsubstantially stereochemically pure product, superior yield and ease ofsynthesis.

EXAMPLE 8 ##STR22##

To a stirred suspension of cyclic HPMPC (1 mmol) was addedN,N'-dicyclohexyl-4-morpholinecarboxamidine (2 mmol) followed by thecorresponding acyloxymethyl chloride (1.5 mmol). The reaction wasstirred for 3 days and the DMF was evaporated under reduced pressure.The crude was purified on a silica gel column (eluted with 5% methanolin methylene chloride) to get the pure cyclic HPMPC derivatives(approximately 30% yield).

We claim:
 1. A compound of structure (1a) ##STR23## wherein *independently designates (S), (R) or (R,S) configuration; B' a 9-purineheterocyclic ring structure;R² is hydrogen, hydroxy, fluorine, chlorine,bromine, amino, or an organic substituent having 1-5 carbon atoms andselected from acyloxy, alkoxy, alkylthio, alkylamino or dialkylamino; A'is OH or A; and A is the residue of an amidate or ester;and the saltsthereof.
 2. The compound of claim 1 wherein the carbon atom * chiralcenter is stereochemically pure as the (S) enantiomer.
 3. The compoundof claim 1 wherein B' is adenine, 2,6 diaminopurine, guanine.
 4. Acompound of structure (Va) ##STR24## wherein * independently designates(S), (R) or (R,S) configuration; A' is OH or A;A is the residue of anamidate or ester; B' is a 9-purine heterocyclic ring structure;and thesalts thereof.
 5. The compound of claim 4 wherein the carbon atom *chiral center is stereochemically pure as the (S) enantiomer.
 6. Thecompound of claim 4 wherein B' is adenine, 2,6 diaminopurine, guanine.